Recombinant Acinetobacter sp. Probable Fe (2+)-trafficking protein (ACIAD0282)

What expression systems are most effective for producing recombinant Acinetobacter Fe(2+)-trafficking proteins?

For the expression of recombinant Acinetobacter Fe(2+)-trafficking proteins, prokaryotic bacterial systems, particularly Escherichia coli, are typically the most effective due to the protein's bacterial origin and relatively simple structure without complex post-translational modifications .

Recommended expression systems based on research findings:

For optimal results with E. coli expression of Fe(2+)-trafficking proteins:

• Select an appropriate E. coli strain (BL21(DE3) is commonly used for recombinant protein expression)

• Optimize codon usage for bacterial expression

• Include appropriate affinity tags for purification (His-tag or GST-tag)

• Consider fusion with endogenous protein sequences to increase yield

What methods are available for purification of recombinant Acinetobacter Fe(2+)-trafficking proteins?

Purification of recombinant Fe(2+)-trafficking proteins typically employs affinity chromatography techniques. Based on established methodologies for similar proteins, the following approaches are recommended:

Affinity Purification Methods:

• Polyhistidine–nickel ion affinity chromatography:

  • Incorporate a 6xHis-tag at the N- or C-terminus of the recombinant protein

  • Use Ni-NTA agarose columns for binding

  • Implement imidazole gradient elution (10-250 mM)

  • Expected purity: >90%

• Glutathione S-transferase (GST)–glutathione affinity:

  • Express protein as a GST fusion

  • Capture on glutathione-agarose

  • Elute with reduced glutathione or cleave with site-specific protease

  • Advantage: May improve solubility of the recombinant protein

Typical purification protocol workflow:

• Cell lysis (sonication or chemical lysis buffer)

• Clarification (centrifugation at 15,000 × g, 30 min, 4°C)

• Affinity chromatography

• Size exclusion chromatography for further purification

• Concentration determination (Bradford or BCA assay)

• Purity assessment via SDS-PAGE (>90% purity is typically achievable)

What experimental designs are optimal for studying Fe(2+)-trafficking protein function in vitro?

When investigating the function of Fe(2+)-trafficking proteins in vitro, several experimental designs can be employed. Based on similar studies with other iron-related proteins in Acinetobacter, the following approaches are recommended:

Experimental Design for Fe(2+)-trafficking Protein Function:

For a full factorial design experiment investigating the effects of iron concentration and oxidative stress on protein function:

Example 2×3 Factorial Design:

• Factor 1: Iron concentration (levels: deficient, normal, excess)

• Factor 2: Oxidative stress (levels: present, absent)

• Dependent variable: Protein activity or binding capacity

• Controls: Wild-type strain, buffer controls

This design requires 6 experimental conditions with a minimum of 3 replicates per condition .

How can researchers track iron trafficking mediated by the Fe(2+)-trafficking protein in real-time experiments?

Real-time tracking of iron trafficking requires sophisticated methodologies. Based on research with similar iron-trafficking systems, the following approaches can be applied:

Real-time Iron Trafficking Monitoring:

• Fusion protein- and intein-based fluorescent labeling strategies:

  • Label Fe(2+)-trafficking protein with fluorescent probes

  • Monitor fluorescence changes upon iron binding/release

  • Different fluorophores can distinguish between cluster types and oxidation states

• Stopped-flow spectrophotometric studies:

  • Monitor rapid kinetics of iron binding and release

  • Assess formation of reaction intermediates

  • Determine rate constants for each step

• Mass spectrometry:

  • Track changes in protein mass corresponding to iron binding/release

  • Monitor protein modifications during iron trafficking

  • Can detect transient intermediates in the trafficking process

Example data from a fluorescence quenching experiment tracking iron transfer:

What is the role of Fe(2+)-trafficking proteins in Acinetobacter baumannii virulence and antibiotic resistance?

Fe(2+)-trafficking proteins likely contribute significantly to A. baumannii pathogenicity based on the critical role of iron in bacterial virulence. Research findings suggest:

• Iron acquisition and virulence correlation:

  • A. baumannii infections show attributable mortality of 14.8-36.5%

  • Iron acquisition systems are essential for establishing infection

  • Fe(2+)-trafficking proteins may facilitate iron utilization during infection

• Iron availability and antibiotic susceptibility:

  • Iron-limiting conditions affect expression of multiple genes, including β-lactamase genes

  • Human serum proteins influence iron uptake gene expression and antibiotic susceptibility

  • Fe(2+)-trafficking proteins may modulate bacterial responses to antibiotics

• Methodological approaches to study this relationship:

  • Gene knockout studies followed by virulence assessment in animal models

  • Growth inhibition assays under iron limitation with/without antibiotics

  • Transcriptome analysis comparing wild-type and mutant strains under infection-like conditions

Iron acquisition proteins represent potential vaccine targets, as demonstrated by studies with outer membrane proteins like Omp22, AbOmpA, and DcaP-like protein .

What techniques can be used to investigate the role of the Fe(2+)-trafficking protein in Fe-S cluster assembly?

The role of Fe(2+)-trafficking proteins in Fe-S cluster assembly can be investigated using several complementary techniques:

Methodological approaches to study Fe-S cluster assembly:

• Fluorescent labeling to track Fe-S cluster transfer:

  • Label Fe-S cluster receptors with fluorophores

  • Monitor fluorescence quenching upon cluster binding

  • Determine kinetics of cluster transfer

• In vitro reconstitution assays:

  • Reconstitute Fe-S cluster assembly systems with purified components

  • Include or exclude the Fe(2+)-trafficking protein

  • Monitor cluster formation spectroscopically

• Protein-protein interaction studies:

  • Use pull-down assays to identify interaction partners

  • Confirm interactions via biolayer interferometry or surface plasmon resonance

  • Map interaction domains through truncation analysis

Sample protocol for Fe-S cluster transfer assay:

• Express and purify recombinant Fe(2+)-trafficking protein

• Label potential Fe-S cluster acceptor proteins with rhodamine

• Prepare [2Fe-2S] cluster-loaded donor proteins

• Mix components and monitor fluorescence quenching over time

• Validate results using mass spectrometry to confirm cluster transfer

How should experiments be designed to assess the impact of iron availability on Fe(2+)-trafficking protein expression and function?

When designing experiments to assess the impact of iron availability on Fe(2+)-trafficking protein expression and function, researchers should consider the following methodological approach:

Full Factorial Experimental Design:

• Independent Variables:

  • Iron concentration (3 levels: iron-depleted, normal, iron-rich)

  • Growth phase (2 levels: exponential, stationary)

  • Stress conditions (2 levels: present, absent)

• Dependent Variables:

  • Protein expression level (qRT-PCR, Western blot)

  • Iron binding activity

  • Bacterial fitness (growth rate)

• Controls:

  • Wild-type strain without manipulation

  • Knockout mutant for the Fe(2+)-trafficking protein

  • Complemented mutant strain

This represents a 3×2×2 full factorial design requiring 12 experimental conditions . For each condition, perform at least three biological replicates and three technical replicates.

Data Table Format for Results:

What are the key considerations for designing a recombinant Fe(2+)-trafficking protein for vaccine development against Acinetobacter baumannii?

Based on recent vaccine development research against A. baumannii, researchers designing recombinant Fe(2+)-trafficking proteins as vaccine candidates should consider:

Key Design Considerations:

• Antigen Selection and Optimization:

  • Identify conserved epitopes across Acinetobacter strains

  • Consider combining Fe(2+)-trafficking protein with other antigens (similar to AbOmpA + DcaP-like protein approach)

  • Evaluate insertion of protein into immunogenic carriers like flagellin

• Expression System Selection:

  • E. coli systems typically yield high amounts of protein

  • Ensure proper folding to maintain conformational epitopes

  • Purify to >90% to minimize adverse reactions

• Immunization Protocol Design:

  • Use appropriate adjuvants (e.g., Alum as used in other A. baumannii vaccines)

  • Implement multiple immunizations (typically three doses)

  • Consider route of administration (subcutaneous, intramuscular, or intranasal)

• Evaluation Methods:

  • Measure specific antibody production (IgG, IgG1, IgG2a/c)

  • Assess cytokine profiles (IL-4, IL-6, IL-17A)

  • Determine bacterial loads in challenge studies

  • Evaluate survival rates against lethal bacterial challenge

Expected Immunological Outcomes Based on Similar Studies:

What methodologies are recommended for studying protein-protein interactions involving the Fe(2+)-trafficking protein?

To investigate protein-protein interactions involving the Fe(2+)-trafficking protein, researchers should employ multiple complementary methodologies:

Recommended Methodological Approaches:

• In vitro Binding Assays:

  • Pull-down assays using recombinant tagged proteins

  • Surface plasmon resonance (SPR) for binding kinetics

  • Isothermal titration calorimetry (ITC) for thermodynamic parameters

  • Biolayer interferometry for real-time binding analysis

• Structural Studies:

  • X-ray crystallography of protein complexes

  • NMR spectroscopy for mapping interaction surfaces

  • Hydrogen-deuterium exchange mass spectrometry to identify binding regions

• In vivo Interaction Studies:

  • Bacterial two-hybrid system

  • Co-immunoprecipitation from bacterial lysates

  • Fluorescence resonance energy transfer (FRET)

  • Proximity ligation assay

Experimental Protocol for Pull-down Assay:

• Express recombinant Fe(2+)-trafficking protein with His-tag

• Immobilize on Ni-NTA resin

• Prepare bacterial lysate containing potential interaction partners

• Incubate immobilized protein with lysate

• Wash extensively

• Elute bound proteins

• Analyze by SDS-PAGE and identify by mass spectrometry

Data Analysis Example:

What are the current challenges in studying Fe(2+)-trafficking proteins in Acinetobacter species?

Researchers face several methodological challenges when studying Fe(2+)-trafficking proteins in Acinetobacter species:

Current Challenges and Methodological Solutions:

• Protein Stability and Solubility Issues:

  • Challenge: Small iron-trafficking proteins may be unstable or form inclusion bodies

  • Solution: Optimize expression conditions (temperature, induction time); use solubility-enhancing tags; consider inclusion body recovery and refolding protocols

• Iron-Binding Specificity Assessment:

  • Challenge: Distinguishing specific from non-specific iron binding

  • Solution: Use multiple negative controls; implement competitive binding assays; validate with multiple techniques

• Functional Redundancy:

  • Challenge: Multiple proteins may have overlapping iron-trafficking functions

  • Solution: Create multiple gene knockouts; perform epistasis analysis; use systems biology approaches to map functional networks

• In vivo Relevance:

  • Challenge: Connecting in vitro observations to physiological significance

  • Solution: Develop animal infection models; use tissue culture systems; implement in vivo imaging techniques for iron trafficking

• Antibiotic Resistance Correlation:

  • Challenge: Establishing causative links between iron trafficking and antibiotic resistance

  • Solution: Design experiments that carefully control for confounding variables; use clinical isolates with varying resistance profiles

What future research directions are most promising for Fe(2+)-trafficking proteins in Acinetobacter?

Based on current knowledge and technological capabilities, several promising research directions emerge:

Future Research Directions:

• Systems Biology Approach:

  • Integration of proteomics, transcriptomics, and metabolomics data

  • Network analysis to position Fe(2+)-trafficking proteins within iron homeostasis systems

  • Computational modeling of iron flux through bacterial systems

• Structural Biology and Drug Development:

  • High-resolution structures of Fe(2+)-trafficking proteins

  • Structure-based drug design targeting these proteins

  • Development of iron-trafficking inhibitors as novel antimicrobials

• Vaccine Development:

  • Assessment of Fe(2+)-trafficking proteins as vaccine antigens

  • Design of multi-epitope vaccines incorporating conserved regions

  • Evaluation in animal models of infection

• Host-Pathogen Interactions:

  • Investigation of how host iron sequestration affects bacterial Fe(2+)-trafficking

  • Examination of Fe(2+)-trafficking protein expression during infection

  • Evaluation of role in biofilm formation and persistence

• Diagnostic Applications:

  • Development of detection methods for Fe(2+)-trafficking proteins

  • Assessment as biomarkers for antibiotic resistance

  • Point-of-care diagnostic tools based on protein detection

Methodological Framework for Future Studies:

To address these future directions, researchers should implement:

• Interdisciplinary collaborations (microbiology, structural biology, immunology)

• Translational research approaches (bench-to-bedside)

• Advanced imaging techniques for real-time tracking

• Machine learning for data integration and prediction

• CRISPR-Cas9 gene editing for precise genetic manipulation